The separation and determination of metoprolol and its metabolites M I (metoprolol acid), M II (2-hydroxy-3-(4-metoxyethylphenoxy)-propanoic acid, M III (a-hydroxymetoprolol) and H 105 (O-desmethylmetoprolol) in human urine by capillary isotachophoresis were investigated. Metoprolol and metabolites M I, M III and H 105 were separated by cationic isotachophoresis in the electrolyte system sodium acetate buffer (pH 5.0) (c,=10 mM) -ß-alanine. Metabolite M II was separated using the anionic electrolyte system histidine hydrochloride buffer (pH 6.0) (cL = 10 mM) -morpholinoethane-sulfonic acid. Endogenous and the possible exogenous compounds were almost totally removed from urine by solid-phase extraction using a SepPak Cls cartridge. The recovery of compounds varied from 84.6 to 95.8%. The linear calibration range was studied for eventual application of the method to real urine samples. Limit of determination was 0.5 mg/ml.